Sufentanil Inhibits Migration of Human Leukocytes Through Human Endothelial Cell Monolayers

Abstract

The interactions between blood and vascular wall cells are essential for understanding pathophysiological processes, e.g., during inflammation. The influence of anesthetics on leukocyte function is well documented. An inhibitory effect of thiopental, midazolam, and ketamine on leukocyte chemotaxis in a Boyden chamber chemotaxis assay (i.e., endothelial cells were not included) has been demonstrated. Little is known, however, about the influence of sufentanil on the inflammatory processes. To reach their targets in the tissue in vivo, leukocytes must interact with endothelial cell monolayers (ECMs). The aim of the current study was to investigate the influence of sufentanil on the migration of leukocytes through an ECM. Human umbilical vein endothelial cells were cultured to achieve a monolayer. Isolated polymorphonuclear leukocytes and ECM were preincubated with different concentrations of sufentanil. The rate of leukocyte migration against the chemotactic protein formyl-methyl-leucyl-phenylalanine was measured (n = 7). Sufentanil significantly reduced the amount of leukocyte migration through ECM to 77% +/- 7.8% (P < 0.05 compared with control). Endothelial cells as well as leukocytes contributed to this effect: treatment of both cell types showed an additive effect. Although lower concentrations showed no effect, high concentrations reduced leukocyte migration through ECM to 61% +/- 7.1%. Implications: Leukocytes play an important role during inflammation, and anesthetics influence leukocyte functions, e.g., respiratory burst or chemotaxis. The effect of sufentanil on transendothelial leukocyte migration has not been investigated. Therefore, we used a migration assay including endothelial cell monolayers. Sufentanil showed a reducing effect on transendothelial leukocyte migration.

(Anesth Analg 1998;87:1181-5)

A knowledge of the interactions between the cells of the vascular wall and blood components is essential for understanding normal vascular biology and pathophysiological processes. In inflammations, leukocytes play an important role in the defense against bacterial infections ^[1]. During migration through endothelial cell monolayers (ECMs), leukocytes undergo morphological changes from rounded, relatively smooth cells to elongated cells ^[2]. The initial phase of leukocyte migration consists of the rolling of neutrophils along the vascular endothelial cells. By binding of their ligands to endothelial cell counterreceptors, activated leukocyte integrins halt the rolling leukocytes and attach them to the vascular endothelium. Adherent leukocytes move to a region between two endothelial cells and migrate into the surrounding tissue ^[3,4].

The influence of anesthetics on leukocytes is well documented ^[5]. Anesthetics can inhibit leukocyte functions ^[6]. In this study, we investigated leukocyte chemotaxis in a Boyden chamber assay (i.e., without endothelial cells). The influence of sufentanil on leukocyte-endothelial cell interactions is poorly understood. Fischer et al. ^[7] described the influence of sufentanil only on brain ECM permeability of ions, without including leukocytes. The aim of the current investigation was to study the influence of sufentanil on the migration of leukocytes through monolayers of endothelial cells in a migration assay simulating physiological conditions.

Methods

Human umbilical venous endothelial cells (HUVEC) were prepared essentially as described by Jaffe et al. ^[8]. Human umbilical cords (n = 7) were collected. Informed consent was obtained, and the study was approved by our institutional review board. HUVEC were freshly isolated with collagenase (Sigma Chemical Company, St. Louis, MO) and washed in medium RPMI-1640 (Gibco, Gaithersburg, MD). Cells were plated at a density of 4 x 10⁶ cells into fibronectin-precoated, 1.2-cm diameter tissue culture wells. Cells were grown in a 37[degree sign]C humidified incubator containing 5% CO₂ in air and were fed approximately every 2 days with fresh medium RPMI-1640 containing 1% L-glutamine, 100 [micro sign]g/mL streptomycin, and 5 [micro sign]g/mL fungizone and were supplemented with 5% fetal calf serum (all reagents from Gibco). After 2 days, cells were passaged with trypsin-EDTA (Gibco), washed with medium twice, and resuspended with medium RPMI-1640 containing the supplement. The HUVEC were seeded on microporous filter membranes (Falcon[trade mark sign]; Becton Dickinson, San Jose, CA) with a pore size of 3.0 [micro sign]m. These inserts were cultured in the incubator until a monolayer was grown as described ^[9-11]. The viability of the ECMs was tested using a life-and-death fluorescence dye calcein-AM (Molecular Probes, Eugene, OR) ^[12] and measured in a fluorescence microscope (Diaphot 100; Nikon, Tokyo, Japan).

Blood was taken from the antecubital vein of seven healthy, nonsmoking test persons aged 25-35 yr without clinical symptoms of inflammation, into Vacuette[trade mark sign] blood collecting tubes (Greiner, Kremsmuenster, Austria) coated with EDTA. Polymorphonuclear leukocytes (PMNL) were isolated using a percoll-ficoll (Pharmacia, Uppsala, Sweden) standard technique ^[13]. The freshly isolated PMNL were washed twice in phosphate-buffered saline (Gibco) and resuspended in RPMI-1640. The cells were counted in the neumayer chamber. Five million PMNL were used per milliliter. The cell viability was tested using the trypan blue exclusion test.

ECMs and/or freshly isolated PMNL were preincubated (30 min) with sufentanil (0.5, 5, and 50 ng/mL) ^[14,15] and used in the double-chamber migration assay. The concentrations used were chosen according to the findings of Freye ^[16].

Our migration assay includes ECMs and is a modification of our cell chemotaxis assay, as previously described ^{[10,11,17-19]}. ECMs were incubated with freshly isolated neutrophils in each well of a 24 multiwell, double-chamber system (Falcon[trade mark sign]). Leukocyte migration was induced by the chemotaxin N-formyl-methyl-leucyl-phenyl-alanine (fMLP; Sigma Chemical Company) in a concentration of 10^-7 mol/L. PMNL and ECMs were incubated with fMLP in the lower system for various time periods. After 3 h, an optimal migratory response was observed. This time point was used in all subsequent experiments. After 3 h of incubation, membranes were detached and removed, together with non-transmigrated cells. The migrated PMNL in the lower chamber were then incubated with the fluorescence dye Calcein-AM (5 [micro sign]mol/L; Molecular Probes) for 30 min at room temperature ^[12]. After the incubation period, the labeled cells were washed and measured in a multiplate fluorometer (Perceptive Biosystems, Hamburg, Germany), and the number of migrated cells was determined by the calculation program provided by the manufacturers. All experiments were performed in duplicate and repeated in seven independent experiments. The migration of untreated PMNL through untreated ECM was used as control and set as 100%.

Standard statistical tests were used for comparison among the different groups (GraphPAD InStat, Version 1.14; GraphPAD Software, San Diego, CA). Analysis of variance was used to compare for within-group and between-group analyses. Values are expressed as means +/- SD. P < 0.05 was considered statistically significant.

Results

The quality of ECMs was characterized by their morphology in the inverse-phase contrast microscope and fluorescence microscope. The cell viability was > 96%. Figure 1 illustrates a migrating cell through a monolayer of endothelial cells from the upper chamber into the lower chamber of the double-chamber migration assay. The amount of untreated leukocyte migration through untreated endothelial cells was used as control and was set as 100%. The total number of migrated PMNL was 1.52 x 10⁶ cells/mL.

Figure 1: Scanning electron micrograph of a cell of the cell line U937 migrating through a monolayer of endothelial cells against the chemoattractant formyl-methyl-leucyl-phenylalanine. The endothelial cells were grown on a microporous membrane (3.0-[micro sign]m pore size) to achieve a monolayer. The formyl-methyl-leucyl-phenylalanine was put in the lower chamber, and the leukocytes were put in the upper chamber. Endothelial cells open their intercellular junctions to allow the migration of an U937 cell into the lower chamber. In the right corner of the image, the rest of a transmigration cell in a microporous membrane is shown.

Clinical relevant concentrations of sufentanil (5 ng/mL) decreased the rate of leukocyte migration through a monolayer of endothelial cells by approximately 20% (P < 0.05 compared with control), when both cell systems-leukocytes and ECMs-were treated. The treatment of leukocytes alone showed a reduction in migration rate by 10% (P < 0.05 compared with control). The treatment of endothelial cells revealed a migration rate of 85% +/- 7.3% (P < 0.05 compared with control).

Sufentanil treatment of both cell types, PMNL and ECM simultaneously, was significant different (P < 0.05) compared with the migration rate of treated PMNL through untreated ECM. The sufentanil treatment of both cell types compared with the migration rate of untreated PMNL through treated ECM was also significant stronger (P < 0.05) (Figure 2).

Figure 2: The influence of a clinically relevant concentration of sufentanil (5 ng/mL) on the migration of polymorphonuclear leukocytes (PMNL) through endothelial cell monolayers (ECM) is shown. The migration rate of untreated leukocyte through untreated ECMs against the chemotaxin formyl-methionyl-leucyl-phenylalanine (10^-7 mol/L) was used as control and set as 100%. The clinically relevant concentration showed significant effects when both cell types (PMNL and ECM) were treated. Sufentanil-pretreated PMNL migration through untreated ECM showed a higher migration rate than untreated PMNL through sufentanil-pretreated ECM. (*P < 0.05 compared with control)

The use of a clinically high concentration (50 ng/mL) of sufentanil reduced leukocyte migration to 77% +/- 7.6% (P < 0.05 compared with control) when only leukocytes were treated, to 70% +/- 8.4% (P < 0.05 compared with control) when only ECMs were treated, and to 61% +/- 7.1% (P < 0.05 compared with control) when both cell types were treated. The pretreatment with a low concentration (0.5 ng/mL) of sufentanil reduced the amount of leukocyte migration to 98% +/- 8.8% (P = not significant [NS] compared with control) when only leukocytes were treated, to 96% +/- 6.3% (P = NS compared with control) when only ECMs were treated, and to 96% +/- 9.1% when both cell types were pretreated (Figure 3 and Figure 4).

Figure 3: The influence of a clinically low dose of sufentanil (0.5 ng/mL) on the migration of polymorphonuclear leukocytes (PMNL) through endothelial cell monolayers (ECM) is shown. The quantity of untreated PMNL through untreated ECMs against the chemotaxin formyl-methionyl-leucyl-phenylalanine (10^-7 mol/L) was used as control and set as 100%. The clinically low concentration showed no significant effect.

Figure 4: The influence of a clinically high dose of sufentanil (50 ng/mL) on the migration of polymorphonuclear leukocytes (PMNL) through endothelial cell monolayers (ECM) is demonstrated. The quantity of untreated leukocyte through untreated ECMs against the chemotaxin formyl-methionyl-leucyl-phenylalanine (10^-7 mol/L) was used as control and set as 100%. The clinically relevant concentration showed significant effect when both cell types (PMNL and ECM) were treated. Using a clinically high concentration showed a significant reducing effect (*P < 0.05 compared with control).

Statistical analysis of the dose-dependence showed significant differences between low versus high and clinically relevant versus high concentrations (P < 0.05) when only PMNL were treated, among all concentrations (P < 0.05) when only ECM were treated, and among all groups (P < 0.05) when both cell types were treated. Additive effects (P < 0.05) could only be shown when the group of treated PMNL was compared with the group of treated PMNL and ECM, but not among the other groups.

Discussion

Leukocytes play a potent role during inflammation. To arrive at the extravascular tissue, leukocytes must migrate from the intravascular space to the surrounding tissue, where they release their granules to combat bacterial infections. Endothelial cells perform a very important function in leukocyte migration ^[1-4].

Drugs can influence leukocyte functions. Previous studies have investigated the influence of different anesthetics on leukocytes only ^[5]. In these studies, the influence of different anesthetics was tested in agarose gel systems. A decrease of leukocyte chemotaxis compared with control has been shown. These in vitro studies did not include endothelial cells, however. In the current model, we investigated the influence of sufentanil on the migration of leukocytes through ECMs. We suggest that the use of more complex in vitro models better describe and mimic the complex in vivo situation.

Sufentanil was identified as a potent inhibitor of leukocyte migration through ECMs. In the current study, sufentanil significantly reduced the migration of leukocytes through ECM to 77% when both cell systems were treated. The opioid receptor antagonist naloxone may have an influence on the effect of sufentanil on leukocyte migration. An article ^[20] on the chemotaxis of human peripheral blood B lymphocytes shows that naloxone has a weak but significant stimulatory effect on the migratory behaviors of B lymphocytes, as well as T lymphocytes. Although substance P-stimulated migration of cells was not affected by naloxone, it prevented the migration of lymphocytes, thus confirming that the migration of lymphocytes is mediated by both opiate- and nonopiate-dependent mechanisms. However, this group investigated chemotaxis, not migration through cell layers. In a previous study, we investigated the influence of the non-steroidal antiinflammatory drug ibuprofen on the migration of leukocytes through ECM using the same assay ^[11]. Data show that ibuprofen can reduce the migration of leukocytes through ECMs by 35%. The assay using ibuprofen allows a functional in vitro validation of a drug that has major effects on leukocytes during inflammation.

Nishina et al. ^[6] demonstrated that different anesthetics, such as thiopental, midazolam, and ketamine, can reduce leukocyte chemotaxis in a microporous filter system. This study avoided the artificial contact of leukocytes with agarose, and this in vitro model is one step closer to the in vivo situation, as the investigators moved from a two-dimensional agarose system to a three-dimensional double-chamber chemotaxis system. In the present study, we developed an in vitro assay for leukocyte migration studies, which includes the partner of leukocytes during the migration process, the endothelial cells.

Until now, it has only been possible to study the influence of drugs on PMNL or on endothelial cells in separate test systems, i.e., not simultaneously. However, after every IV injection, both leukocytes and endothelial cells are in direct contact with the drug. Hence, this investigation on sufentanil effects may be a somewhat more physiological model because it studies drug influence on leukocytes as well as on endothelial cells. In this test assay, we found a significant suppressive effect on leukocyte migration when both cell systems were drug-treated. These data support the clinical observations that anesthetics may influence the immune system by reducing leukocyte recruitment.

The design of the migration assay used also allows the separate treatment of leukocytes and ECM. Therefore, we investigated the quantity of migrated sufentanil-treated leukocytes through untreated ECMs, as well as untreated leukocytes through sufentanil-treated ECMs. A dose-dependence could be shown in all groups except low versus clinically relevant concentrations in the PMNL group. The sufentanil treatment of both cell types-leukocytes and endothelial cells-showed an additive effect compared with PMNL.

In conclusion, sufentanil reduces leukocyte migration through ECMs. Leukocytes, as well as endothelial cells, seem to be influenced. The treatment of both cell types showed an additive effect.

We thank Dr. Zwoelfer (Department of Anesthesiology, Hospital Privatklinik Doebling, Vienna, Austria) and the midwives (Department of Gynecology, Hospital Privatklinik Doebling, Vienna, Austria) for collecting of the umbilical cords. We also thank Professor H. G. Kress, MD, Head of the Department, and Professor M. Zimpfer, MD, Director of the Clinic of Anesthesiology, for allowing us to conduct part of the study in their research laboratories.